Helical polycarbodiimide polymers and associated imaging, diagnostic, and therapeutic methods

ABSTRACT

Described herein are suspensions of helical polycarbodiimide polymers that ‘cloak’ nanotubes, thereby effecting control over nanotube emission, providing a new mechanism of environmental responsivity, and enabling precise control over sub-cellular localization. The helical polycarbodiimide polymers described herein are water soluble, easily modifiable, and have unique architectures that facilitate their application in radiopharmaceutical delivery and imaging methods, in therapeutics and therapeutic delivery methods, and their use as sensors—both in conjunction with carbon nanotubes, and without nanotubes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of, and incorporatesherein by reference in its entirety, U.S. Provisional Patent ApplicationNo. 62/038,235.

GOVERNMENT SUPPORT

This work was supported by National Institutes of Health grantDP2-HD07569.

FIELD OF THE INVENTION

This invention relates generally to compositions comprisingpolycarbodiimide polymers, and related imaging, diagnostic, andtherapeutic methods.

BACKGROUND

Carbon nanotubes have several features which demonstrate their potentialfor biomedical applications including cellular sensing and imaging. Forexample, carbon nanotubes can be metallic or semiconducting depending ontheir structure, which is due to the symmetry and unique electronicstructure of graphene. Thus, the electronic structure and diameter ofthe carbon nanotube will determine the spectral characteristics seen inabsorption, fluorescence, Raman scattering, etc. Moreover, theenvironmental sensitivity and intrinsic photostability of single-walledcarbon nanotubes (SWCNTs) in the near-infrared wavelength range (ca. 900nm-1600 nm) demonstrates the potential of biomedical applications.However, such uses require the ability to simultaneously modulatenanotube fluorescence and to biocompatibly derivatize the nanotubesurface using noncovalent methods.

Both covalent and non-covalent functionalization methods can be used tosolubilize carbon nanotubes for adaptation to biomedical applications.Non-covalent functionalization of SWCNTs preserves both the optical andstructural properties of SWCNTs in solution. Nanotubes can beencapsulated in various amphiphilic polymers. Biopolymers such as singlestranded DNA (ssDNA), peptides, or proteins, and synthetic polymers,such as polyfluorenes, polycarbazoles, aryleneethynylene polymers,polyethylene glycol (PEG) derivatives, and dextran-based polymers havebeen investigated for materials, and biological applications. However,encapsulation with biopolymers can produce higher background signal(e.g., DNA can produce high oxidation current and subsequently higherbackground currents) or provide inadequate control of coating thenanotube and therefore affect optical response (e.g., proteindenaturation in unfavorable conditions). Moreover, the above-mentionedsynthetic polymers do not provide controllable and/or tunableproperties, which limit the ability to measure (e.g., via imaging) thekinetics of dynamic self-assembly and disassembly and translocation ofphotoluminescent nanotubes into live cell nuclei.

Therefore, there is a need to better adapt carbon nanotubes forbiomedical applications, such as cellular imaging and sensing, thatprovides control over nanotube emission, environmental responsivity,precise control over sub-cellular localization, ordered surfacecoverage, and systematic modulation of nanotube optical properties.

SUMMARY OF INVENTION

Described herein are suspensions of helical polycarbodiimide polymersthat ‘cloak’ nanotubes, thereby effecting control over nanotubeemission, providing a new mechanism of environmental responsivity, andenabling precise control over sub-cellular localization. The helicalpolycarbodiimide polymers described herein are water soluble, easilymodifiable, and have unique architectures that facilitate theirapplication in radiopharmaceutical delivery and imaging methods, intherapeutics and therapeutic delivery methods, and their use assensors—both in conjunction with carbon nanotubes, and withoutnanotubes.

For example, the helical polycarbodiimide polymers can be modified withradionuclides or radionuclide-chelating agents. Experiments performedwith these polymers—for example, DOTA-modified polymer with multiplechelation sites for Lutetium-177—demonstrate rapid clearance and loworgan update, especially in the kidneys.

The helical polycarbodiimide polymers can also deliver molecules andincrease drug binding affinity via multivalency, lending to their use astherapeutics and in therapeutic delivery, for example, opiate-polymerconjugates that provide long-term analgesic effects, as well astreatment of cancer, atherosclerosis, skin disorders, infectiousdiseases, and other diseases. Due to the semi-rigidity of the polymer,more binding sites are accessible, compared with polymers having aglobular form. Furthermore, the helical polymer lengths are short andvery controllable, allowing for rapid clearance if desired.

Moreover, the helical polymers described herein are demonstrated toencapsulate single-walled carbon nanotubes, which are used asfluorescent sensors for in vitro, ex vivo, and in vivo applications. Thepolymers provide both sensitivity to specific, desired bioanalytes, anddirect/target the sensors to specific locations in the cell and body.Polymer-nanotube constructs are shown that provide nuclear, cytosolic,and extracellular localization. Moreover, a stable polymer-nanotubesensor is presented for in vitro and in vivo redox potentialmeasurements.

In one aspect, the invention is directed to a suspension ofhelical-polymer-encapsulated carbon nanotubes, wherein the helicalpolymer is a polycarbodiimide. In certain embodiments, the carbonnanotubes are single-walled carbon nanotubes (SWCNTs). In certainembodiments, the helical polymer comprises a clickable polymer scaffold.In certain embodiments, the polycarbodiimide comprises one or moremonomeric species selected from the group consisting of

In certain embodiments, the suspension is an aqueous suspension. Incertain embodiments, at least a plurality of thehelical-polymer-encapsulated carbon nanotubes in the suspension are invan der Waals contact at a center-to-center distance between adjacentnanotubes sufficient to exhibit inter-nanotube Förster resonance energytransfer (INFRET). In certain embodiments, the center-to-center distanceis from 1 nm to 4 nm. In certain embodiments, the dispersedhelical-polymer-encapsulated carbon nanotubes in van der Waals contactare not irreversibly bound.

In certain embodiments, the suspension comprises (i) a first set ofnanotubes each encapsulated by a helical polymer having at least a firstsubstituent functional group (e.g., a primary amine), and (ii) a secondset of nanotubes each encapsulated by a helical polymer having at leasta second substituent functional group (e.g., a carboxylic acid), whereinthe first substituent functional group and the second substituentfunctional group imbue the first and second sets of encapsulatednanotubes with sufficiently strong coulombic attraction to each other toform reversible fluorescent aggregates in the suspension.

In certain embodiments, the helical polymer comprises functional sidechains. In certain embodiments, the functional side chains comprise oneor more members selected from the group consisting of a primary amine, acarboxylic acid, a guanidine group, an oligoethylene glycol, amethoxy-polyethylene glycol (PEG), a hydroxyl-PEG, a folic acid, atrimethoprim, a peptide, an alkyne peptide, an adenosine triphosphate(ATP) peptide, and an opioid. In certain embodiments, the helicalpolymer comprises one or more aromatic groups incorporated in itsmonomer substituents. In certain embodiments, the one or more aromaticgroups promote multi-valent π-π interactions between the polymer and thegraphitic sidewall of the carbon nanotubes.

In certain embodiments, the functional side chains comprise a targetinggroup (e.g., an organelle targeting group, a protein targeting group, apolysaccharide targeting group, or a targeting group for anotherbiological structure). In certain embodiments, a biomolecular imagingprobe and/or sensor comprises the suspension.

In another aspect, the invention is directed to an imaging methodcomprising: administering the suspension to a biological sample (e.g.,in vitro, ex vivo, or in vivo, e.g., wherein the biological sample is asubject); exposing the biological sample comprising the administeredsuspension to excitation light (e.g., near-infrared excitation light);and detecting light emitted by suspension or fluorescent aggregatesformed by one or more components of the suspension (e.g., detectinglight by inter-nanotube Förster resonance energy transfer (INFRET)) inthe biological sample).

In certain embodiments, the imaging method comprises disrupting thefluorescent aggregates (e.g., wherein disrupting the fluorescentaggregates is performed by administering an agent) to reverse theemission of light.

In certain embodiments, the imaging method comprises alternating betweencycles of light emission and no light emission by re-aggregating anddisrupting, respectively, the fluorescent aggregates (e.g., for highresolution biomolecular imaging).

In certain embodiments, the detecting step comprises obtaining images ofcellular nuclei of the biological sample.

In another aspect, the invention is directed to a method of treating adisease or disorder (e.g., cervical, pancreatic, or skin cancer), themethod comprising administering the suspension to a subject, wherein thefunctional side chains of the helical polymer comprises a therapeutic.

In certain embodiments, the therapeutic comprises an opiate.

In another aspect, the invention is directed to a method for pretargetedradioimmunotherapy (PRIT), the method comprising administering apolycarbodiimide functionalized with an antibody, labeled with aradionuclide (e.g., wherein administering the labeled and functionalizedpolycarbodiimide delivers cytotoxic radiation to a target cell of thesubject).

In certain embodiments, the radionuclide comprises a metallic lanthanide(e.g., yttrium or lutetium). In certain embodiments, the radionuclide isattached to the polycarbodiimide via a chelator (e.g.,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ordesferoxamine (DFO)). In certain embodiments, the radionuclide comprises⁸⁹Zr.

In certain embodiments, the suspension is a stable suspension (e.g.,stable in aqueous solution or in serum).

In another aspect, the invention is directed to a polycarbodiimidepolymer having a helical conformation comprising one or more functionalgroups (e.g., functional side chains).

In certain embodiments, the one or more functional groups comprise atleast one member selected from the group consisting of a primary amine,a carboxylic acid, a guanidine group, an oligoethylene glycol, amethoxy-PEG, a hydroxyl-PEG, a folic acid, a DOTA(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) or anotherchelator or complexing agent, a trimethoprim, a perphenazine, a peptide,an alkyne peptide, an ATP peptide, and an opioid.

In certain embodiments, the polymer is labeled with a radionuclide. Incertain embodiments, the radionuclide comprises a metallic lanthanide(e.g., yttrium or lutetium). In certain embodiments, the radionuclide isattached to the polycarbodiimide via a chelator (e.g.,1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) ordeferoxamine (DFO)). In certain embodiments, the radionuclide comprises⁸⁹Zr.

In certain embodiments, the polymer is functionalized with an antibody.

In certain embodiments, the polymer comprises a fluorophore. In certainembodiments, fluorophore is an infrared (IR) dye.

In certain embodiments, the polymer comprises a therapeutic (e.g., anopiate, e.g., octreotide).

In certain embodiments, the polymer comprises multimeric targetinggroups (e.g., for receptors in cancer cells).

In another aspect, the invention is directed to a method of treating adisease or disorder, the method comprising administering the polymer toa subject, wherein the polymer is functionalized with a therapeutic.

In another aspect, the invention is directed to a method forradiotherapy (e.g., PRIT), the method comprising administering thepolymer to a subject, wherein the polymer is functionalized with aradionuclide.

In another aspect, the invention is directed to an imaging methodcomprising: administering the polymer of any one of claims 27 to 37 to abiological sample (e.g., wherein the administering is in vitro, ex vivo,or in vivo, e.g., wherein the biological sample is a subject); exposingthe biological sample comprising the administered suspension toexcitation light (e.g., near-infrared excitation light); and detectingelectromagnetic radiation emitted by at least one of the one or morefunctional groups of the polymer.

In certain embodiments, the method further comprises exposing thebiological sample to excitation light (e.g., near-infrared excitationlight) prior to (and/or concurrent with) the detecting step, wherein thedetecting step comprises detecting emitted fluorescent light.

In another aspect, the invention is directed to ahelical-polymer-encapsulated carbon nanotube, wherein thehelical-polymer is a polycarbodiimide.

In certain embodiments, the carbon nanotube is in a solid form (e.g.,powdered or adhered to a surface) and capable of forming a stablesuspension in solution (e.g., aqueous solution, e.g., serum).

In another aspect, the invention is directed to a method of utilizing asensor to detect and/or monitor the presence of and/or concentration ofone or more analytes (e.g., pathogens or other bioanalytes) in a sample,the method comprising: administering and/or contacting the suspensionand/or the polymer to/with the sample; following the administeringand/or contacting step, allowing one or more components of theadministered and/or contacted suspension and/or polymer to accumulate inthe sample, wherein the one or more components exhibit a detectablesensitivity to the one or more analytes to be assayed; and following theaccumulation, obtaining a measurement (e.g., a 1D, 2D, or 3Dmeasurement, map, or image (e.g., positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) image)) indicative ofthe presence and/or concentration of the one or more analytes in thesample.

In certain embodiments, the sample is a biological sample, and the oneor more analytes is/are bioanalyte(s).

In certain embodiments, the method comprises allowing the one or morecomponents of the administered and/or contacted suspension and/orpolymer to accumulate in an extracellular location of the biologicalsample.

In certain embodiments, the method comprises allowing the one or morecomponents of the administered/contacted suspension and/or polymer toaccumulate in an intracellular location (e.g., in cellular nuclei and/orin the cytosol) of the biological sample.

In certain embodiments, the biological sample is an in vitro, ex vivo,or in vivo sample (e.g., wherein the biological sample is a subject).

In certain embodiments, the administering and/or contacting stepcomprises administering and/or contacting a device (e.g., a chip,microneedle delivery device, or transdermal patch) comprising thesuspension and/or the polymer to/with the sample (e.g., whereincontacting comprises embedding, adhering, injecting, or placing thedevice into or onto the sample).

In certain embodiments, the functional side chains of the helicalpolymer comprise a radiolabel, and wherein the measurement is ameasurement of radiation emitted by the radiolabel. In certainembodiments, functional side chains comprise a complex of1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with ametallic lanthanide (e.g., yttrium or lutetium).

In another aspect, the invention is directed to a method of utilizing asensor to detect and/or monitor a redox potential in a sample, themethod comprising: administering and/or contacting the suspension of anyone of claims 1 to 14 to/with the sample; optionally, following theadministering and/or contacting step, allowing one or more components ofthe administered and/or contacted suspension to accumulate in the sample(e.g., further comprising, absorbing a quantity of the sample into atest strip or isolating a quantity of the sample for redox measurement;e.g., further comprising, contacting the isolated sample with thesuspension, rather than the one or more components of the suspensionmoving into the sample); and exposing the one or more components of theadministered and/or contacted suspension to an applied voltage, andmeasuring the resulting redox potential (e.g., a 1D, 2D, or 3Dmeasurement or map) of the sample.

In certain embodiments, the sample is a biological sample, and themeasured reduction potential is in the range from −150 millivolts to−400 millivolts.

In certain embodiments, the method further comprises allowing the one ormore components of the administered/contacted suspension to accumulatein an extracellular location of the biological sample.

In certain embodiments, the method further comprises allowing the one ormore components of the administered/contacted suspension to accumulatein an intracellular location (e.g., a cellular nuclei and/or a cytosol)of the biological sample.

In certain embodiments, the functional side chains of the helicalpolymer comprise an organelle targeting group.

In certain embodiments, the biological sample is an in vitro, ex vivo,or in vivo sample.

In certain embodiments, the administering and/or contacting stepcomprises administering or contacting a device (e.g., a chip,microneedle delivery device, or transdermal patch) comprising thesuspension to/with the sample (e.g., wherein contacting comprisesembedding, adhering, injecting, or placing the device into or onto thesample).

In certain embodiments, the biological sample is skin and wherein theone or more components of the administered and/or contacted suspensionare delivered to and embedded within an epidermal layer of the skin(e.g., further comprising monitoring an extracellular redox potentialover time).

In another aspect, the invention is directed to a kit for use in aradiopharmacy setting, the kit comprising: at least one container,wherein the container has a type selected from an ampule, a vial, acartridge, a reservoir, a lyo-ject, or a pre-filled syringe; thepolymer, wherein the molecular weight (e.g., weight average molecularweight or number average molecular weight) is from 5 kDa to 75 kDa(e.g., from 10 kDa to 50 kD, e.g., from 15 kDa to 30 kDa); at least onedisposable size exclusion column; and at least one disposable filter,wherein the at least one container holds (e.g., contains) the polymer.

DEFINITIONS

In order for the present disclosure to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

In this application, the use of “or” means “and/or” unless statedotherwise. As used in this application, the term “comprise” andvariations of the term, such as “comprising” and “comprises,” are notintended to exclude other additives, components, integers or steps. Asused in this application, the terms “about” and “approximately” are usedas equivalents. Any numerals used in this application with or withoutabout/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

“Administration”: The term “administration” refers to introducing asubstance into a subject. In general, any route of administration may beutilized including, for example, parenteral (e.g., intravenous), oral,topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal,rectal, nasal, introduction into the cerebrospinal fluid, orinstillation into body compartments. In some embodiments, administrationis oral. Additionally or alternatively, in some embodiments,administration is parenteral. In some embodiments, administration isintravenous.

“Analyte”: As used herein, the term “analyte” broadly refers to anysubstance to be analyzed, detected, measured, or quantified. Examples ofanalytes include, but are not limited to, proteins, peptides, hormones,haptens, antigens, antibodies, receptors, enzymes, nucleic acids,polysaccharides, chemicals, polymers, pathogens, toxins, organic drugs,inorganic drugs, cells, tissues, microorganisms, viruses, bacteria,fungi, algae, parasites, allergens, pollutants, and combinationsthereof.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe materials that do not elicit a substantial detrimental responsein vivo. In certain embodiments, the materials are “biocompatible” ifthey are not toxic to cells. In certain embodiments, materials are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and/or their administration in vivo does notinduce inflammation or other such adverse effects. In certainembodiments, materials are biodegradable.

“Biodegradable”: As used herein, “biodegradable” materials are thosethat, when introduced into cells, are broken down by cellular machinery(e.g., enzymatic degradation) or by hydrolysis into components thatcells can either reuse or dispose of without significant toxic effectson the cells. In certain embodiments, components generated by breakdownof a biodegradable material do not induce inflammation and/or otheradverse effects in vivo. In some embodiments, biodegradable materialsare enzymatically broken down. Alternatively or additionally, in someembodiments, biodegradable materials are broken down by hydrolysis. Insome embodiments, biodegradable polymeric materials break down intotheir component polymers. In some embodiments, breakdown ofbiodegradable materials (including, for example, biodegradable polymericmaterials) includes hydrolysis of ester bonds. In some embodiments,breakdown of materials (including, for example, biodegradable polymericmaterials) includes cleavage of urethane linkages.

“Carrier”: As used herein, “carrier” refers to a diluent, adjuvant,excipient, or vehicle with which the compound is administered. Suchpharmaceutical carriers can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,such as peanut oil, soybean oil, mineral oil, sesame oil and the like.Water or aqueous solution saline solutions and aqueous dextrose andglycerol solutions are preferably employed as carriers, particularly forinjectable solutions. Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin.

“Detector”: As used herein, the term “detector” includes any detector ofelectromagnetic radiation including, but not limited to, CCD cameras,photodiodes, optical sensors, and infrared detectors.

“Functionalization”: As used herein, the term “functionalization” refersto any process of modifying a material by bringing physical, chemical orbiological characteristics different from the ones originally found onthe material. Typically, functionalization involves introducingfunctional groups to the material. As used herein, functional groups arespecific groups of atoms within molecules that are responsible for thecharacteristic chemical reactions of those molecules. As used herein,functional groups include both chemical (e.g., ester, carboxylate,alkyl) and biological groups (e.g., adapter, or linker sequences).

In some embodiments, click reactive groups are used (for ‘clickchemistry’). Examples of click reactive groups include the following:alkyne, azide, thiol (sulfydryl), alkene, acrylate, oxime, maliemide,NHS (N-hydroxysuccinimide), amine (primary amine, secondary amine,tertiary amine, and/or quarternary ammonium), phenyl, benzyl, hydroxyl,carbonyl, aldehyde, carbonate, carboxylate, carboxyl, ester, methoxy,hydroperoxy, peroxy, ether, hemiacetal, hemiketal, acetal, ketal,orthoester, orthocarbonate ester, amide, carboxyamide, imine (primaryketimine, secondary ketamine, primary aldimine, secondary aldimine),imide, azo (diimide), cyanate (cyanate or isocyanate), nitrate, nitrile,isonitrile, nitrite (nitrosooxy group), nitro, nitroso, pyridyl,sulfide, disulfide, sulfinyl, sulfonyl, sulfino, sulfo, thiocyanate,isothiocyanate, caronothioyl, thione, thial, phosphine, phosphono,phosphate, phosphodiester, borono, boronate, bornino, borinate, halo,fluoro, chloro, bromo, and/or iodo moieties.

“Image”: The term “image”, as used herein, is understood to mean avisual display or any data representation that may be interpreted forvisual display. For example, a three-dimensional image may include adataset of values of a given quantity that varies in three spatialdimensions. A three-dimensional image (e.g., a three-dimensional datarepresentation) may be displayed in two-dimensions (e.g., on atwo-dimensional screen, or on a two-dimensional printout). The term“image” may refer, for example, to an optical image, an x-ray image, animage generated by: positron emission tomography (PET), magneticresonance, (MR) single photon emission computed tomography (SPECT),and/or ultrasound, and any combination of these.

“Radiolabel”: As used herein, “radiolabel” refers to a moiety comprisinga radioactive isotope of at least one element. Exemplary suitableradiolabels include but are not limited to those described herein. Insome embodiments, a radiolabel is one used in positron emissiontomography (PET). In some embodiments, a radiolabel is one used insingle-photon emission computed tomography (SPECT). In some embodiments,radioisotopes comprise ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Ga, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm,¹⁷⁷Lu, ⁶⁷Cu, ¹²³I, ¹²⁴I, ¹²⁵I, ¹¹C, ¹3N, ¹⁵O, ¹⁸F, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm,¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁴⁹Pm, ⁹⁰Y, ²¹³Bi, ¹⁰³Pd, ¹⁰⁹Pd, ¹⁵⁹Gd, ¹⁴⁰La, ¹⁹⁸Au,¹⁹⁹Au, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶⁷Cu, ¹⁰⁵Rh, ¹¹¹Ag, ⁸⁹Zr, ²²⁵Ac, and¹⁹²Ir.

“Sample”: The term “sample” refers to a volume or mass obtained,provided, and/or subjected to analysis. In some embodiments, a sample isor comprises a tissue sample, cell sample, a fluid sample, and the like.In some embodiments, a sample is taken from (or is) a subject (e.g., ahuman or animal subject). In some embodiments, a tissue sample is orcomprises brain, hair (including roots), buccal swabs, blood, saliva,semen, muscle, or from any internal organs, or cancer, precancerous, ortumor cells associated with any one of these. A fluid may be, but is notlimited to, urine, blood, ascites, pleural fluid, spinal fluid, and thelike. A body tissue can include, but is not limited to, brain, skin,muscle, endometrial, uterine, and cervical tissue or cancer,precancerous, or tumor cells associated with any one of these. In anembodiment, a body tissue is brain tissue or a brain tumor or cancer.Those of ordinary skill in the art will appreciate that, in someembodiments, a “sample” is a “primary sample” in that it is obtainedfrom a source (e.g., a subject); in some embodiments, a “sample” is theresult of processing of a primary sample, for example to remove certainpotentially contaminating components and/or to isolate or purify certaincomponents of interest.

“Subject”: As used herein, the term “subject” includes humans andmammals (e.g., mice, rats, pigs, cats, dogs, and horses). In manyembodiments, subjects are be mammals, particularly primates, especiallyhumans. In some embodiments, subjects are livestock such as cattle,sheep, goats, cows, swine, and the like; poultry such as chickens,ducks, geese, turkeys, and the like; and domesticated animalsparticularly pets such as dogs and cats. In some embodiments (e.g.,particularly in research contexts) subject mammals will be, for example,rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine suchas inbred pigs and the like.

“Therapeutic agent”: As used herein, the phrase “therapeutic agent”refers to any agent that has a therapeutic effect and/or elicits adesired biological and/or pharmacological effect, when administered to asubject.

“Treatment”: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a substance that partiallyor completely alleviates, ameliorates, relives, inhibits, delays onsetof, reduces severity of, and/or reduces incidence of one or moresymptoms, features, and/or causes of a particular disease, disorder,and/or condition. Such treatment may be of a subject who does notexhibit signs of the relevant disease, disorder and/or condition and/orof a subject who exhibits only early signs of the disease, disorder,and/or condition. Alternatively or additionally, such treatment may beof a subject who exhibits one or more established signs of the relevantdisease, disorder and/or condition. In some embodiments, treatment maybe of a subject who has been diagnosed as suffering from the relevantdisease, disorder, and/or condition. In some embodiments, treatment maybe of a subject known to have one or more susceptibility factors thatare statistically correlated with increased risk of development of therelevant disease, disorder, and/or condition.

Drawings are presented herein for illustration purposes, not forlimitation.

BRIEF DESCRIPTION OF DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofthe present disclosure will become more apparent and better understoodby referring to the following description taken in conjunction with theaccompanying drawings, in which:

FIGS. 1A-1B depict preparation of polycarbodiimide-SWCNT complexes.

FIG. 1A shows synthesis of polycarbodiimide polymers (Poly-1-8).

FIG. 1B depicts an exemplary scheme showing preparation of thepolycarbodiimide-SWCNT aqueous suspension.

FIGS. 2A-2F show optical and morphological properties ofpolycarbodiimide-SWCNTs.

FIG. 2A shows vis-nIR absorption spectra.

FIG. 2B shows nIR emission spectra of polycarbodiimide-SWCNTs (16 mg/Lnanotubes) excited at 659 nm.

FIG. 2C shows center wavelengths of nanotube emission peaks collectedfrom photoluminescence excitation/emission profiles ofpolycarbodiimide-SWCNTs and surfactant suspended SWCNTs.

FIG. 2D shows atomic force micrograph of Amine-Poly-8-SWCNT complexesshowing periodic banding along the nanotube surface.

FIG. 2E shows a magnified AFM image of a single Amine-Poly-8-SWCNTcomplex.

FIG. 2F shows a height profile of a single complex denoted by the whitearrow in FIG. 2E.

FIGS. 3A-3B show two-dimensional near-infrared photoluminescenceexcitation/emission (PLE) plots showing normalized emission intensityfrom polycarbodiimide-SWCNTs and surfactant suspended SWCNTs (SDS andSDC) as a function of excitation wavelength.

FIGS. 4A-C show atomic force microscopy and transmission electronmicroscopy images of polycarbodiimide-SWCNTs.

FIG. 4A shows atomic force microscopy height images ofAmine-Poly-6-SWCNT.

FIG. 4B shows atomic force microscopy height images ofAmine-Poly-8-SWCNTs.

FIG. 4C shows atomic force microscopy height images of transmissionelectron microscopy images of polycarbodiimide-SWCNTs.

FIGS. 5A-5G show reversible inter-nanotube Förster resonance energytransfer (INFRET) in polycarbodiimide-SWCNTs.

FIG. 5A shows a schematic representation of the INFRET process and itsreversal upon addition of amine-functionalized polycarbodiimide.

FIG. 5B shows photoluminescence excitation-emission (PLE) map ofAmine-Poly-6-SWCNTs, Carboxy-Poly-7-SWCNTs, mixture ofAmine-Poly-6-SWCNTs and Carboxy-Poly-7-SWCNTs, and the mixture aftersubsequent addition of amine-polymer.

FIG. 5C shows a nanotube (n, m) species-dependent PL intensity changeupon initiating INFRET.

FIG. 5D shows a nanotube (n, m) species-dependent PL intensity changeupon INFRET reversal.

FIG. 5E shows individual spectra acquired during a time courseacquisition of INFRET kinetic data. Intensity was normalized to the areaunder the curve.

FIG. 5F shows INFRET dynamics show a monotonic relative PL intensityincrease in small bandgap nanotubes (Peaks 4 and 5) and simultaneousrelative PL intensity decrease in large bandgap nanotubes (Peaks 1-3).

FIG. 5G shows INFRET ratio, plotted using Peak 5 as the acceptor andPeak 1 as the donor. The final data point was acquired after initiatingINFRET reversal using amine-polymer.

FIG. 6A shows near-infrared images of polycarbodiimide-SWCNTsimmobilized on glass surfaces showing discrete fluorescent nanotubes(left and middle panels). Dilute solutions were placed on 35 mm glassbottom petri dishes for 10 seconds and excess solution was removed priorto imaging the nanotubes on the surface. The right panel shows nIRfluorescent aggregates of polycarbodiimide-SWCNTs after mixing the twonanotube complexes in solution. Carboxy-Poly-7-SWCNT was added toAmine-Poly-6-SWCNT (1:1 ratio) and left to stand for 10 sec; excesssolution was removed from the surface prior to imaging.

FIG. 6B shows height projection near-infrared image ofpolycarbodiimide-SWCNTs aggregates, generated by 3D deconvolution of astack of images acquired in 10 μm Z-steps.

FIG. 6C shows PL Intensity change upon initiating and reversingself-assembly of Amine-Poly-7-SWCNTs and Carboxy-Poly-8-SWCNTs.

FIGS. 7A and 7A-1 show that the kinetic data from FIG. 5F was fit with alogistic function (of the form

$y = {\frac{{A\; 1} - {A\; 2}}{1 + \left( \frac{x}{x\; 0} \right)^{p}} + {A\; 2}}$

to obtain the parameters in the accompanying table.

FIG. 7B shows the curves of peaks P1 and P5 fit the classical solutionsfor the reactant and product, respectively, in a consecutive series offirst order chemical reactions.

FIGS. 8A-8C show near infrared (nIR) fluorescence images of living HeLacells incubated with polycarbodiimide-SWCNTs.

FIG. 8A, comprising panels A1-A5, shows cells incubated with specifiedpolymernanotube complexes at 37° C. for 18 h. Panel A5, for example, isa micrograph of cells incubated at 4° C. for 4 h. A 730 nm laser wasused for excitation and light was collected over 900 nm-1400 nm.

FIG. 8B, comprising panels B1-B5, shows combined nIR fluorescence andbrightfield images of living HeLa cells.

FIG. 8C, comprising panels C1-C2, shows brightfield, nIR fluorescenceand Hoechst nuclear stain of live HeLa cells incubated in the presenceof Amine-Poly-8-SWCNTs and Guanidine-Poly-4-SWCNTs, respectively.

FIGS. 9A-9B show near-infrared fluorescence images of live HeLa cellsincubated with Guanidine-Poly-4-SWCNTs. Near-infrared—bright-fieldoverlays illustrate nuclear localization.

FIG. 10 shows viability assays on HeLa cells incubated withpolycarbodiimide-SWCNT complexes. HeLa cells were incubated in thepresence of specified polymer-nanotube complexes in a 35 mm petri dishfor 24 hours before imaging. Tali™ Image-Based Cytometer was used tomeasure cell viability test performed using Tali™ viability kit-Deadcell red (Invitrogen) following manufacturer's protocol.

FIG. 11 shows near-infrared and brightfield overlay micrographs of humanskin, showing the lack of penetration of polycarbodiimide-SWCNTs,regardless of functionalization. Polycarbodiimide-SWCNTs deposits mainlyin the stratum corneum layer of the skin. Skin was acquired from humanpatients after Moh's surgery and immediately incubated withpolycarbodiimide-SWCNTs on the surface of the skin specimen. Skinsections without nanotube treatment were used as control.

FIGS. 12A-12C show a synthesis scheme and molecular structures forhelical polycarbodiimide polymers as described herein.

FIGS. 13A-13B depict schematics showing the manufacturing of anano-sensor dispersion, according to an illustrative embodiment.

FIG. 13A shows representative scheme of sensor fabrication into finalsensor dispersion (top) and examples of applications for the nano-sensor(bottom).

FIG. 13B shows example of sensor modification allowing specifictargeting to sub-cellular locations.

FIGS. 14A-14B show illustrative data from a nano-sensor dispersion.

FIG. 14A shows data showing the linear response of a nano-sensor todecreasing reduction potential mediated in a buffered aqueous biologicalsolution of cysteine and ascorbate as major redox couples. Each markedline represents a unqiuely responding chiral nanotube within the samesensor population.

FIG. 14B shows data showing different chiralities of the sensor can acttogether to form a ratiometric fluorescence response, thereby allowingquantitative measurement.

FIG. 15 shows the activities in liver, spleen, and kidney ranged from1-2% ID/g suggesting that clearance from blood primarily occurred fromthe kidney, as typically hepatic/RES clearance is slow in comparison andshows high and prolonged uptake/retention in the liver and spleen (e.g.liposomes).

FIG. 16 shows representative images of polycarbodiimide polymers withopiate substituent groups.

FIG. 17 shows synthesis of polymers: DFO-conjugated polymer DFO-JBP1 andpolymer without DFO, mPEG-JBP2. DFO-JBP1 was synthesized to measureradiolabeling efficiency, in vitro stability, and in vivo performance ofpolycarbodiimide polymers without targeting ligands. mPEG-JBP2 serves asa negative control to ensure ligand specific binding of radiometal,89Zr. Polymer probe design integrates targeting ligands, radiometalchelators, and solubilizing groups in a single polymer chain.

FIGS. 18A-18C show radiolabeling of Polymers and Serum Stability Tests.

FIG. 18A shows ⁸⁹Zr labeling of DFO-JBP1 showed greater than 99%radiolabeling.

FIG. 18B depicts that a control polymer mPEG-JBP2 showed negligiblelabeling (less than 3%). Radiolabeling was performed at room temperaturefor 30 minutes.

FIG. 18C depicts that ⁸⁹Zr-labeled polymer, or ⁸⁹Zr-DFO-JBP1, showed 99%stability in human serum at 37° C. over seven days.

FIGS. 19A-19B show PET imaging and biodistribution of ⁸⁹Zr-RadiolabeledPolymer, ⁸⁹Zr-DFO-JBP1.

FIG. 19A shows coronal PET images of ⁸⁹Zr-DFO-JBP1. Healthy BALB/c mice(n=2) were intravenously administered ⁸⁹Zr-DFO-JBP1 (˜10 mg, ˜100 μCi,250 mL saline) and imaged at 2 h, 24 h, and 72 h post injection. Initialtime points showed high activities in the blood, whereas at 72 h p.imost of the activities were detected in the liver.

FIG. 19B shows ex vivo biodistribution of ⁸⁹Zr-DFO-JBP1 in select majororgans. At 96 h p.i, the mice (n=2) were sacrificed, organs wereextracted, and radioactivities and organ weights were measured andexpressed as the % injected dose per gram of tissue.

FIG. 20 shows synthesis of urea derivatives, monomers, and correspondingpolymers as described herein.

FIGS. 21A-21B show the FTIR spectra of polymers, Poly-1 and Poly-2,respectively.

FIG. 22 shows azide compounds 5-7 that were synthesized andcharacterized in certain embodiments as described herein.

The features and advantages of the present disclosure will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings, in which like reference charactersidentify corresponding elements throughout. In the drawings, likereference numbers generally indicate identical, functionally similar,and/or structurally similar elements.

DETAILED DESCRIPTION

Throughout the description, where compositions are described as having,including, or comprising specific components, or where methods aredescribed as having, including, or comprising specific steps, it iscontemplated that, additionally, there are compositions of the presentinvention that consist essentially of, or consist of, the recitedcomponents, and that there are methods according to the presentinvention that consist essentially of, or consist of, the recitedprocessing steps.

It should be understood that the order of steps or order for performingcertain action is immaterial so long as the invention remains operable.Moreover, two or more steps or actions may be conducted simultaneously.

The mention herein of any publication, for example, in the Backgroundsection, is not an admission that the publication serves as prior artwith respect to any of the claims presented herein. The Backgroundsection is presented for purposes of clarity and is not meant as adescription of prior art with respect to any claim.

Described herein are suspensions of helical polycarbodiimide polymersthat ‘cloak’ nanotubes, thereby effecting control over nanotubeemission, providing a new mechanism of environmental responsivity, andenabling precise control over sub-cellular localization. The helicalpolycarbodiimide polymers described herein are water soluble, easilymodifiable, and have unique architectures that facilitate theirapplication in radiopharmaceutical delivery and imaging methods, intherapeutics and therapeutic delivery methods, and their use assensors—both in conjunction with carbon nanotubes, and withoutnanotubes.

For example, the helical polycarbodiimide polymers can be modified withradionuclides or radionuclide-chelating agents. Experiments performedwith these polymers—for example, DOTA-modified polymer with multiplechelation sites for Lutetium-177—demonstrate rapid clearance and loworgan update, especially in the kidneys.

The helical polycarbodiimide polymers can also deliver molecules andincrease drug binding affinity via multivalency, lending to their use astherapeutics and in therapeutic delivery, for example, opiate-polymerconjugates that provide long-term analgesic effects, as well astreatment of cancer, atherosclerosis, skin disorders, infectiousdiseases, and other diseases. Due to the semi-rigidity of the polymer,more binding sites are accessible, compared with polymers having aglobular form. Furthermore, the helical polymer lengths are short andvery controllable, allowing for rapid clearance if desired.

Moreover, the helical polymers described herein are demonstrated toencapsulate single-walled carbon nanotubes, which are used asfluorescent sensors for in vitro, ex vivo, and in vivo applications. Thepolymers provide both sensitivity to specific, desired bioanalytes, anddirect/target the sensors to specific locations in the cell and body.Polymer-nanotube constructs are shown that provide nuclear, cytosolic,and extracellular localization. Moreover, a stable polymer-nanotubesensor is presented for in vitro and in vivo redox potentialmeasurements.

In addition, the helical polymers described herein are demonstrated tobe radiolabeled and serve as multimodal targeted molecular imagingprobes for early cancer, such as pancreatic cancer, detection. Thepolymers integrate multimeric targeting ligands for receptors in cancercells to achieve high tumor specific uptake and retention, containmultiple chelators to chelate multiple radiometals for enhanced specificactivity and quantitative PET imaging, and allow tunable hydrophilicitythrough minimal structural changes to increase plasma stability, prolongprobe circulation in vivo, improve pharmacokinetics, and reduceimmunogenicity.

Example 1 Helical Polycarbodiimide Cloaking of Carbon Nanotubes

In the examples described herein, a platform of helical polycarbodiimidepolymers was synthesized to ‘cloak’ the nanotubes which affected controlover nanotube emission, provided a new mechanism of environmentalresponsivity, and enabled precise control over sub-cellularlocalization. The helical polymers exhibited ordered surface coverage onthe nanotubes, allowed systematic modulation of nanotube opticalproperties, and produced up to 12-fold differences in photoluminescenceefficiency. The polymers facilitated controllable and reversibleinter-nanotube Förster resonance energy transfer, allowing kineticmeasurements of dynamic self-assembly and disassembly. Tailoredpolycarbodiimide substituent groups also enabled sub-cellular targetingfor imaging, including stable translocation of photoluminescentnanotubes within live cell nuclei.

Synthetic helical polymers mimic the basic structural motifs of vitalbiomolecules such as DNA and peptides. The functions of helical polymersdepend on conformation, chain flexibility, and on the array offunctional moieties along the polymer backbone. Polycarbodiimides aresynthetic helical polymers with tunable properties. Polycarbodiimideregioregularity is shown through ¹⁵N-isotope labeling studiesdemonstrating precise control of the polymer microstructure andpost-modification in a regioregular polycarbodiimide, resulting in apolymer chain with a regular array of functional side chains.

A modular polycarbodiimide polymer system is presented herein thatcloaks nanotubes in repeating chemical functional units and suspendspristine nanotubes in aqueous solutions. Alkyne polycarbodiimides(Poly-1 and Poly-2) were synthesized and organic azides weresubsequently coupled to terminal alkyne groups in these polymers viaCu(I) catalyzed alkyne-azide cycloaddition, as depicted in FIG. 1A. Sidechains in these polymers such as primary amines, carboxylic acids,guanidine groups, and oligoethylene glycols were incorporated to mimicside chains in polylysines, polyglutamic acids, and polyarginines, andto increase water solubility. Additionally, aromatic groups wereincorporated in each monomer substituent 3 and 4 (FIG. 1A) to promotemulti-valent π-π interactions between the polymer and the graphiticsidewall of SWCNTs. Raw SWCNTs (Unidym, HiPCO) were sonicated in thepresence of a polycarbodiimide from the library (Poly 3-8) to renderthem soluble in an aqueous solution. The insoluble materials werepelleted via ultracentrifugation and removed, yielding a dark aqueoussupernatant (FIG. 1B). Excess free polymer was then removed from thesuspensions by centrifugal filtration. The aqueous suspensions werestable under ambient conditions for several months, with no visibleaggregation.

Polycarbodiimide-SWCNT complexes were characterized by absorptionspectroscopy in the vis-nIR region. Absorption spectra of allpolycarbodiimide-SWCNT complexes in FIG. 2A shows characteristic E₂₂ andE₁₁ transition features of semi-conducting SWCNTs. Sharp, discrete peaksin the absorption spectra are indicative of well-dispersed nanotubes.The photoluminescence efficiencies of the polymer-nanotube complexesvaried with the encapsulating polymer. FIG. 2B shows thephotoluminescence intensities from polycarbodiimide-SWCNTs differing upto 12-fold, depending on the polymer substituent functional group aswell as the polymer microstructure. Such trends are similar to findingsreported for DNA-encapsulated SWCNTs.

Two-dimensional photoluminescence excitation/emission (PLE) spectroscopywas conducted on polycarbodiimide-SWCNTs by recording emission spectraupon varying the excitation wavelength, as described below. Fourteendistinct nanotube species detected in 2D PLE plots onpolycarbodimide-SWCNT complexes (FIGS. 3A and 3B) were assigned (n, m)chirality indices. Excitation and emission wavelength maxima, collectedfrom the PLE plots fell within a narrow range which was red-shiftedrelative to surfactant-suspended SWCNT emission (FIG. 2C).

Atomic force microscopy (AFM) and transmission electron microscopy (TEM)were conducted to characterize polycarbodiimide-SWCNT morphology (FIGS.2D-2F, FIGS. 4A-4C). Images of Amine-Poly-8-SWCNTs, deposited and driedon freshly cleaved mica surface, show a distinct, periodic bandingpattern along the nanotube surface. The patterns exhibit a spacing of˜20 nm along the nanotube axis and band heights up to ˜0.8 nm-0.5 nmabove the surface of the nanotubes. Without having to be bound bytheory, these observations, coupled with the long-term stability of thepolymer-nanotube suspension, suggest a uniform conformation of thesearomatic polymers along the SWCNTs. These AFM micrographs are comparableto those from DNA encapsulated SWCNTs, where a regular banding patternof DNA strands with a pitch of 14-20 nm along the nanotubes have beenreported. Without having to be bound by theory, based on this regularpattern and the similarity to the pattern in DNA-SWCNTs which arepredicted (by all atom molecular dynamics (MD) simulations) to helicallywrap nanotubes via the π-π interactions, the polymer also likelyhelically-encapsulates the nanotubes.

Förster resonance energy transfer (FRET), also described as excitonenergy transfer (EET) in SWCNTs, has been observed between adjacentsemiconducting nanotubes in van der Waals contact wherein large band gapdonors transfer energy to smaller band gap acceptors. In small bundles,a center to center distance of 1-4 nm between nanotubes was shown tooptimize energy transfer in SWCNTs. With a functionally-diverse set ofpolymer-SWCNTs in hand, the possibility of inter-nanotube Försterresonance energy transfer (INFRET) events betweenindividually-encapsulated nanotubes in aqueous solutions wasinvestigated. FIG. 5A is a schematic representation of the process. FIG.5B shows 2D PLE plots of two oppositely-charged polymer-nanotubecomplexes (zeta potential values 67.93±2.73 mV for Amine-Poly-6-SWCNTsand −62.93±1.28 mV for Carboxy-Poly-7-SWCNTs) and the resulting mixture.Complexes were chosen to take advantage of strong coulombic attractionbetween basic primary amine groups and acidic carboxylic acid groups tobring nanotubes encapsulated in corresponding polymers into a favorabledistance for INFRET, without creating irreversibly formed van der Waalsbundles. Mixing the two polymer-SWCNT complexes resulted in fluorescentaggregates (FIGS. 6A-6B). Overall emission in PLE measurement decreasedlikely due to quenching induced by metallic SWCNTs in aggregates.However, the emission from smaller band gap SWCNTs increased withrespect to that of large band gap SWCNTs (FIG. 5B). Extra peaks appearedin the short wavelength excitation/long wavelength emission range, asignature of energy transfer. The relative intensity increase was foundto exhibit an (n, m) dependence which was virtually monotonic withemission wavelength (FIG. 5C). Excess amine-functionalizedpolycarbodiimide was later introduced to disrupt aggregation. Additionof the free polymer resulted in a recovery of the original relative PLintensities concomitant with the disappearance of large aggregates (FIG.5B, FIG. 6C). Plotting the net recovery of (n, m) intensities showed asemi-monotonic trend with emission wavelength and apparent mod-dependentbehavior (FIG. 5D).

Real-time measurements of INFRET dynamics illustrate that the process isspontaneous, controllable, and reversible. Upon mixing theaforementioned oppositely-charged nanotubes, the fluorescence exhibiteda monotonic decrease in PL intensities from large bandgap nanotubes(Peaks 1-3, FIG. 5E) and simultaneous relative increase from smallbandgap nanotubes (Peaks 4 and 5, FIG. 5E). The relative fluorescenceintensities of each peak, plotted over time, illustrate the INFRETdynamics between large and small band gap nanotubes (FIG. 5F). Eachintensity-time curve was fit with the logistic function to obtain thetime for half-maximal intensity change (FIGS. 7A and 7A-1), due to thisfunction's use in the approximation of protein aggregation kinetics.However, the kinetics of Peak 1 and Peak 5 fit well as the reactant andthe final product in a series of first order forward reactions,respectively (FIG. 7B). The first order behavior suggests that thelarger bandgap nanotubes within Peak 1 act almost purely as energydonors and the smaller bandgap nanotubes within Peak 5 as energyacceptors. After 110 minutes, amine-functionalized polycarbodiimidepolymer (0.5 mg/mL) was added and gently mixed, resulting in anear-instantaneous reversal of INFRET back to the initial ratio. Usingthe above information, The INFRET ratio, plotted as I_(a)/(I_(a)+I_(d))where I_(a) is the acceptor intensity and I_(d) is the donor intensity,was obtained using Peak 5 as the acceptor and Peak 1 as the donor (FIG.5G).

The finding demonstrates FRET produced between nanotubes not containedwithin an irreversible bundle, but rather employing coulombic attractionbetween polymers permitted spontaneous forward and directedreversibility. Therefore, the described compositions are useful, forexample, for the measurement of dynamic processes.

The biological fate of the polycarbodiimide-cloaked carbon nanotubes wasfound to depend almost completely on the encapsulating polymersubstituent groups. Cellular interactions of polycarbodiimide-SWCNTs inhuman cervical cancer cells (HeLa cells) were investigated.Polycarbodiimide-nanotube complexes exhibited substituent-dependentuptake and localization into specific sub-cellular spaces (FIGS. 8A-8C).The cellular uptake was highly diminished in the case of polyethyleneglycol polymer pendant groups (FIG. 8A, panel A1), comparable to lack ofcellular uptake of PEGylated gold nanoparticles.

Upon internalization, sub-cellular distribution of nanotubes wasdictated by the nature of the encapsulating polymer substituent groups.The anionic Carboxy-Poly-7-SWCNTs accumulated in perinuclear areas (FIG.8A, panel A2), resembling the cellular distribution of DNA-encapsulatednanotubes. The two amine-functionalized polymer-nanotube constructsexhibited sub-cellular distribution profiles where a small fractionlocalized within the nucleus and the majority situated elsewhere withinthe cell (FIGS. 8A, panel A3 and 8C, panel C1). Nanotubes encapsulatedin polymers with guanidine side chains localized almost completelywithin the nuclear region (FIGS. 8A, panel A4 and 8C, panel C2, FIGS. 9Aand 9B) as confirmed by co-localization of nIR fluorescent nanotubeswith Hoechst nuclear dye (Molecular Probes). The nuclear translocationof cargoes by polyarginines and their derivatives has been attributed tothe presence of guanidine moieties. Multiple copies of adjacentguanidine side chains in Guanidine-Poly-4 presumably mimickedpolyarginine side chains, delivering their encapsulated nanotube cargosinto the nuclei. The energy-dependence of complex internalization wasconfirmed by the lack of noticeable cellular uptake upon incubation at4° C. (FIG. 8A, panel A5). Under experimental conditions, thepolymer-SWCNTs posed no obvious toxicity to cells (viability greaterthan 90%, FIG. 10).

Micrographs were obtained to determine whether the variable surfacechemistries of polycarbodiimide-SWCNTs allow penetration of intact humanskin tissue topical exposure. The micrographs show accumulation of alltested polymer-nanotube complexes on the stratum corneum, the outermostlayer of the skin, without evident penetration (FIG. 11).

Thus, the experiments show that non-covalent functionalization of SWCNTsthrough encapsulation in designed helical polycarbodiimides forms watersoluble, well-dispersed, and nIR fluorescent nanotubes that are stableunder ambient conditions. The polymers, used in certain embodiments asdescribed herein, demonstrated controllable, reversible inter-nanotubeFRET, enabling a mechanism for switchable biomolecular probes andsensors. The polymers, as used in certain embodiments as describedherein, also demonstrate a system substituent-dependent sub-cellularlocalization of nanotubes, including stable localization in cell nuclei.

Example 2 Synthesis of Helical Polycarbodiimide Polymers, for Use inTherapeutic and Diagnostic Applications

A synthesis scheme and molecular structures for helical polycarbodiimidepolymers described herein are presented in FIGS. 12A-12C.

For synthesis of urea derivatives, a primary amine compound (RNH₂) (1.0equiv) was diluted in anhydrous dichloromethane and added to anisocyanate compound (RNCO) (1.2 equiv) in dichloromethane, stirred atlow temperature, and kept cold in an ice bath. The reaction mixture wasstirred at room temperature or refluxed overnight until the completionof the reaction. The solvent was removed in a rotary evaporator andcrude white solid was purified by recrystallization in ethanol at 4° C.and dried to obtain white crystalline solid.

For synthesis of carbodiimide monomers, triethyl amine (2.5 equiv) wasadded to a suspension of dibromotriphenylphosphorane (1.2 mol equiv) indichloromethane at low temperature and the reaction mixture was stirredat low temperature under inert atmosphere for 5 minutes. A ureaderivative (1.0 equiv) was added to the reaction mixture and stirreduntil completion. The dehydration of the urea derivative intocarbodiimide monomer was monitored by the formation of a very strongFTIR signal at ˜2120-2140 cm⁻¹. Upon completion of the reaction, hexanewas added to precipitate side products. The monomer compound was thenextracted from the solid by hexanes. Crude monomer was further purifiedby column chromatography on silica gel using ethyl acetate:hexanes (1:2)and dried under reduced pressure to obtain a carbodiimide monomer as acolorless oil.

The catalyst was synthesized and characterized as described in Tang, H.;Boyle, P.; Novak, B., Chiroptical switching polyguanidine synthesized byhelix-sense-selective polymerization using[(R)-3,3′-dibromo-2,2′-binaphthoxy](di-tert-butoxy)titanium(IV)catalyst. Journal of the American Chemical Society 2005, 2136-2142.

The polymers were synthesized following the procedure described inBudhathoki-Uprety, J.; Novak, B., Synthesis of Alkyne-FunctionalizedHelical Polycarbodiimides and their Ligation to Small Molecules using‘Click’ and Sonogashira Reactions. Macromolecules 2011, 44 (15),5947-5954. Briefly, the catalyst, either neat or dissolved in chloroform(0.2 mL per 500 mg monomer) was added to the monomer at room temperatureand under inert atmosphere. The reaction mixture turned to dark red andsolidified to an orange red solid. The polymerization process wasmonitored in FTIR by disappearance of IR signals from carbodiimide(˜2140-2120 cm⁻¹) and formation of new IR absorption at ˜1620-1640 cm⁻¹of the polymer backbone. Upon completion of the polymerization (ca. 24h), the solid was dissolved in chloroform, precipitated in methanol,separated, and dried to obtain light yellow solid.

Organic azides were coupled to the polymers via ‘click’ chemistry. Tothe stirring polymer solution in tetrahydrofuran under inert atmosphere,azide compound (1.0 mol equiv per alkyne unit), triethyl amine or DBU(6.0 mol equiv per alkyne unit) and CuI (10 mol %) were added. Thereaction mixture was stirred overnight under an argon atmosphere.Coupling of small molecules azides to alkyne side chains in polymers wasmonitored by FTIR analysis. Upon completion of the reaction, theresulting polymer was washed with THF and/or diethyl ether, separated byfiltration and dried under reduced pressure. Basic polymers wereacidified with a few drops of dilute HCl and carboxylic acidfunctionalized polymer was treated with a few drops of saturatedsolution of NaHCO₃ to increase water solubility. Acidic and basicpolymer solutions were then filtered through centrifugal filters (AmiconUltracel®, MWCO 3K Da, Merck Millipore Ltd) to remove residual smallmolecules and washed with water until free from free acid or base astested with litmus paper. The polymers were then used to suspend SWCNTs.

Example 3 Nanoscale Sensors for Quantitative Redox Potential Measurement

Reduction potential (or Redox) is a physical concept used to measure thetendency of chemical compounds (couples) to transfer electrons during areaction, and by extension the chemical potential energy in a system orcouple. The direction, regulation, and capacity for cellular activitydepends upon the state of these redox reactions, quantifiable with anelectric potential voltage, for phenomena as diverse as energyproduction, biosynthesis, gene expression, signaling and detoxification.Redox Biology currently remains largely qualitative. Recent linkagebetween perturbations in redox state and cancerous transformation, cellgrowth and division, cell viability, drug efficacy, and numerouspathologies have increased interest in quantitative Redox Biology.

In certain embodiments, the compositions described herein allow for aSingle-Walled Carbon Nanotube (SWCNT) based optical sensor for thispurpose. Current art is not capable of measuring this parameter inliving samples or using materials that have commercialization capabilityfor wide spread use across diverse markets.

In certain embodiments, this sensor utilizes the optical fluorescenceproperties of SWCNTs dispersed with a unique Polycarbodiimide (JB-2-18or JB-2-104) which enables the aqueous dispersed SWCNT to assume anelectronic structure responsive to voltage change in the physiologicallyrelevant redox potential range of approximately −150 millivolts to −400millivolts. The ability of SWCNTs to sensitively respond to appliedvoltage has been tested and modeled in non-aqueous systems. The sensordirectly measures this parameter within aqueous systems usingnon-invasive near infrared fluorescence emission.

The link between redox state and disease makes accurate measurement ofredox increasingly important, both as a direct mechanism of pathology oras an indirect biomarker for screening. Human pathologies linked toaberrant redox state at either the mechanistic or biomarker levelinclude, but are not limited to, sepsis, renal disease, cardiovasculardisease, cancer carcinogenesis and therapy, inflammation, Alzheimer'sdisease, Parkinson's disease, Traumatic Brain Injury, Autism,atherosclerosis, Schizophrenia and Bipolar Disorder, Metabolicdisorders, wounding and tissue regeneration, skin and cellular aging,skin damage and carcinogenesis, and gastrointestinal inflammation anddisease. All basic research applications on various diseases can benefitgreatly from a commercial tool for the measurement of redox potentialfor discovery of mechanisms, biomarkers, and screening therapies.

Current and future biomarkers relating disease to aberrant redoxpotentials or abnormal redox couples must be measured and detected inclinical chemistry laboratories for patient diagnostics. In certainembodiments, this sensor is useful as a measurement tool for diagnosingpatients in clinical settings. The sensors are non-degradable andrequire no special storage, reducing the need for upkeep of traditionalmachinery and biochemical tools like antibodies and enzymes.

Given increasing evidence for the role of oxidants and redox couples inskin aging, skin cancer, and skin damage, in certain embodiments, anavailable microneedle delivery process delivers nano-sensors to, andembeds within, the epidermal layers of the skin for constant monitoringof extracellular redox. For example, current delivery platforms arecommercially available from 3M Company. Measurement of redox viafluorescence emission is obtained with light directed at the skin atwavelengths innocuous to tissue. This gives consumers and physicians theability to track skin exposure and damage from oxidants/chemicals andradiation, tracking of possible pre-cancerous abnormalities, and/ordetermination of post-cancer treatment efficacy and progression.Furthermore, because epidermal skin is constantly shed in 2-3 weekcycles, this sensor is temporary, therefore affording no personal risk.

In other embodiments, these nano-sized sensors are fabricated on chipplatforms and integrated with technology thereby giving consumers theability to measure the redox potential of consumer products or solutionsin daily life in a mobile fashion where, for example, this sensor isintegrated into smart phone platforms as an additional plug-inapplication and attachment. Furthermore, physicians can similarly usethis technology as a quick tool to analyze fluids. The redox potentialof fluids, for example, changes markedly if pathogens are present andproliferating. Given that many consumer products on the market areformulated with strict chemical constituents or various solutions (skincare products, foods) the redox potential measured can indicate thequality or harm of a product and its stability over time.

Industrial and process engineering sectors require measurement of redoxpotential to monitor solution quality of dyes, foods, chemicals,microorganism growth media, and cosmetics. For example, fermentation ofyeast for industrial scale alcoholic beverage production requiresquality control including redox measurement of samples at various stagesof development. Similar process control exists for other industries.Most of these measurements currently require large expensive probes andmachinery. Furthermore, the volume of sample taken from production tomeasure can be significant. In certain embodiments, nano-sensorsdescribed herein, developed in the redox range of interest, can be usedto continuously monitor this measurement in real time and decreasesignificantly the volume needed for measurement.

In an experimental example, a nano-sensor was fabricated by mixingSingle-Walled Carbon Nanotubes (SWCNTs), available from variousdistributors, in a 1:10 ratio by weight with polycarbodiimide,solubilized in water. The mixture is then probe tip sonicated at 30%amplitude and approximately 4-5 Watts for 20 minutes. This resultingdispersion is then worked up: ultracentrifugation for 30 minutes,cut-off filtration using a benchtop centrifuge

2-3 times for 6 minutes each, re-dilution in water, and a final benchtopcentrifugation at maximum force for 20 minutes. The resulting dispersionis ready for use, but may be subjected to an additional optional step.

Using the sensor merely requires addition of the final dispersion intothe medium to be measured, or into the cell culture media for incubationand uptake via cellular processes. The concentration of the sensordispersion can be gathered by taking the absorption of the solution at630 nm, and dividing the valley by a known coefficient, for a result inmg/L.

In certain embodiments, detection of fluorescence emission requires anexcitation source, preferentially a laser, at a wavelength near theresonant absorption of the proper nano-sensor chirality. Unlike organicfluorophores, nanotubes absorb off-resonant light; therefore, manylasers commonly used today are compatible. As with other optical tools,an appropriate filter set and infrared camera are used to detect theemission signal.

Analysis of data is similar to analysis of other fluorescence datacurrently in use. Information with nanotubes is usually gathered asspectra where differences, intensity, and chromatic shifting in peaksare analyzed, or via tracking of individual sensors in microscopy,whereby spectral and spatial information is collected from samples (i.e.cells).

FIGS. 13A-13B depict schematics showing the manufacturing of anano-sensor dispersion, according to an illustrative embodiment. FIGS.14A-14B show illustrative data from a nano-sensor dispersion. As shownin FIG. 14A, the data show the linear response of a nano-sensor todecreasing reduction potential, mediated in a buffered aqueousbiological solution of cysteine and ascorbate as major redox couples.Each marked line represents a uniquely responding chiral nanotube withinthe same sensor population. As shown in FIG. 14B, the data shows thatdifferent chiralities of the sensor act together to form a ratiometricfluorescence response, thereby allowing quantitative measurement.

Example 4 Helical Polymers for Pretargeted Radioimmunotherapy

Recently Orcutt et al. reported a novel scFv antibody (“C825”) with pMaffinity for low molecular weight (MW)1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)complexes with various metallic lanthanides including yttrium (Y) andlutetium (Lu) (Orcutt K D, Slusarczyk A L, Cieslewicz M, Ruiz-Yi B,Bhushan K R, Frangioni J V, et al. Engineering an antibody withpicomolar affinity to DOTA chelates of multiple radionuclides forpretargeted radioimmunotherapy and imaging. Nuclear medicine andbiology. 2011; 38:223-33.). Specifically intended for pretargetedradioimmunotherapy (PRIT), Orcutt and colleagues also preparedbi-specific antibodies having the format IgG-scFv which incorporated thesequences for C825, as well as those for IgG antibodies with highaffinity and specificity for cancer cell-surface targets (e.g.carcinoembryonic (CEA) antigen). During PRIT in vivo with the IgG-C825constructs, the IgG-C825 was initially administered and ample time wasallowed for accumulation at the tumor, followed with a clearing agent toremove freely circulating IgG-C825. In the last step, a low-MWDOTA-hapten would be administered, which would be recognized byprelocalized IgG-C825. In order to obtain optimum therapeutic index, theDOTA-hapten would show rapid blood clearance via the renal route, aswell as low non-specific uptake and retention in normal tissues,including those associated with the reticuloendothelial system (RES).With rapid clearance and minimum retention in tissues, the residencetime of the radioactivity is minimized, thus reducing the absorbed doseto those tissues (and consequently limiting the maximum tolerated dose).The biodistribution and clearance properties of various DOTA-haptenshave been described by Orcutt and colleagues.

Experiments described herein show that the DOTA-Bn-polymers can beradiolabeled with Lu-177 with radiochemical purities sufficient for invivo biodistribution studies. Radioactivity in blood was 0.131±0.125%ID/g at 2 hr post-injection, indicating rapid clearance fromcirculation. As shown in FIG. 15, the activities in liver, spleen, andkidney ranged from 1-2% ID/g suggesting that clearance from bloodprimarily occurred from the kidney, as typically hepatic/RES clearanceis slow in comparison and shows high and prolonged uptake/retention inthe liver and spleen (e.g. liposomes). In all other organs assayed(including s.c. human tumor xenograft, heart, lungs, stomach, small andlarge intestines, muscle, and bone), the radioactivity concentrationswere consistently less than 1% ID/g at 2 hr p.i. suggesting low uptakeand retention in those tissues.

These DOTA-Bn-polymers are useful not only because of their favorableclearance and biodistribution properties, but also because of theirmultivalent design (i.e. greater than 1 DOTA/polymer). It has beenreported that there is improvement in overall tumor uptake duringpretargeted radioimmunotherapy with radioactive bivalent “janus” haptensin comparison with monovalent haptens. The DOTA/polymer stoichiometryallows for addition of a radioactive DOTA-metal complex or for whichC825 does not show pM affinity (e.g. copper or actinium), followed bycold lutetium or yttrium metal. This allows for the non-radioactiveY/Lu-DOTA present on the polymer to serve as an affinity handle forantibody recognition and capture. Pretargeting GD2-positive solid tumorsin mice with antibody-streptavidin fusions has been shown. However,instead of using radioactive biotin as the targeting hapten, tworadiolabeled biotinylated peptides and radiolabeled and biotinylatedbovine serum albumin can also be effectively used. Thus, small peptidesand proteins can be targeted via biotinylation and the pretargetingstrategy.

As described herein, the DOTA-Bn-polymer was supplied as a light yellowdry powder. A stock solution was prepared by adding 200 μL of 0.5 Mammonium acetate pH 5.3 to 5 mg of DOTA-Bn-polymer (25 mg/mL). Theresulting solution appeared as a suspension, and stored at −20° C. Toradiolabel with Lu-177, 20 μL of the stock was added to an acid-washedplastic Eppendorf tube, followed with 10 μL of DMSO and an additional100 μL 0.5 M ammonium acetate pH 5.3 (e.g., to solubilize theDOTA-Bn-polymer). To this solution, 11.55 mCi (427.4 MBq) of Lu-177 wasadded (as ¹⁷⁷LuCl₃ in 0.05 N HCl, specific activity: 170 MBq/nmol;Perkin Elmer), the reaction was vortexed to mix, and the reaction wasincubated at 80° C. for 90 min. To chelate any remaining free metal, 15μL of 50 mM DTPA pH 7 was added, and the reaction was allowed toincubate for an additional 10 min at room temperature. To separate the¹⁷⁷Lu-DOTA-Bn-polymer from ¹⁷⁷Lu-DTPA, the crude reaction was applied toa PD-10 desalting column (Sephadex G-25; greater than 5000 M_(r); GEHealthcare) that was pre-equilibrated with saline for injection, andeluted with additional saline. According to the manufacturer, the voidvolume is ˜2.5 mL, and the total column volume is 8.3 mL. Theradioactivity concentrations in each elution fraction as well as thecolumn itself were determined by assay in a Capintec CRC-25R dosecalibrator using the manufacturer's recommended settings for theisotope.

TABLE 1 Fraction Volume Lu-177 activity 1 reaction (~160 μL), 0 2 1 mL 03 1 mL 66.8 4 1 mL 436 5 0.5 mL 470 6 0.5 mL 674 7 0.5 mL 910 8 0.5 mL1025

After collection of the 8^(th) fraction (total load+elution=˜5.2 mL),the column was assayed in the dose calibrator (7.68 mCi, 66% of appliedradioactivity). The radiochemical purity (RCP) of fractions 4 and 5 wereassayed by thin-layer chromatography (Baker-flex Silica Gel IB-F;elution solvent 1/1 methanol/10% sodium acetate (aq);¹⁷⁷Lu-DOTA-Bn-polymer R_(f)=0.125-0.15, ¹⁷⁷Lu-DTPA R_(f)˜1). The plateradioactivity was assayed using a Bioscan radioTLC scanner. Fraction 4showed a single peak with an R_(f)=0.125, while fraction 5 showed 2peaks (major peak: 86.1% of total radioactivity on plate R_(f)=0.125;minor peak: 13.9% of plate radioactivity R_(f)=0.15). Fraction 3 wasassumed to have the same radiochemical purity as fraction 4. Fractions3, 4, and 5 were combined for injection (overall RCP ˜90% of radioactivespecies with R_(f)=0.125). For injection, doses comprising of 82.4-90.4μCi of ¹⁷⁷Lu-activity (presumably as ¹⁷⁷Lu-DOTA-Bn-polymer) wereformulated in 200 μL final volume of saline.

Two groups (n=5/group) of athymic nu/nu female nude mice (6-8 weeks old;Harlan Sprague Dawley) bearing IMR32-Luc subcutaneous xenografts in thelower flank (average size 1.47 g or 1.39 cm³ assuming a density of 1.05g/mL) were injected intravenously with ¹⁷⁷Lu-DOTA-Bn-polymer using thetail vein. One of the groups was sacrificed 2 hr post-injection (p.i.)and the other at 24 hr p.i. for ex vivo assay of radioactivebiodistribution. Mice were euthanized, and tumor and selected organswere harvested, weighed, and radioassayed by gamma scintillationcounting (Perkin Elmer Wallac Wizard 3”). Count rates were converted toactivities using a system calibration factor, decay corrected andnormalized to the administered activity, and expressed as percentinjected dose per gram (% ID/g).

Example 5 Opiate Polycarbodiimide Conjugates for Drug Delivery andPeripheral Analgesia

Two polycarbodiimide polymers containing opiate substituent groups weresynthesized.

The first (P32) was found to translocate rapidly into the nuclei ofcertain cells. The polymer is able to translocate fully into the nucleuswithin three hours after administration in vitro. The construct is ableto transport large materials, including carbon nanotubes, into thenucleus. Representative images are shown in FIG. 16.

The P33 construct was constructed to function as a peripherally actingopiate analgesic with a preferable side-effect profile over morphine(low euphoria, respiratory depression, physical dependence, addiction),and long-lasting analgesic efficacy above morphine. In vitro and in vivodata of both P32 and P33 polymers are presented in Table 2 below.

TABLE 2 Binding in Opioid transfected CHO cell lines Analgesia in CD1mice given subcutaneously Ki (nM) MOR DOR KOR 6TM/E11 ED50 (mg/kg) P321.25 ± 0.34 13.11 ± 2.88  0.56 ± 0.01 12.67 ± 2.1 not analgesic P33 3.45± 0.55 5.88 ± 0.79 0.85 ± 0.18 10.75 ± 0.9 10 Terminology: MOR = Muopiate receptor DOR = Delta opiate receptor KOR = Kappa opiate receptor6TM/E11 = 6 transmembrane domain E11 splice variant of MOR

Example 6 Radiolabeled Polymers as Multimodal Targeted Molecular ImagingProbes for Early Pancreatic Cancer Detection

The present disclosure describes dual-modal positron emission tomography(PET) and fluorescent imaging agents with multimeric targeting ligandsfor enhanced receptor binding and multiple radiometal chelators forimproved signal and high-resolution imaging. Molecular imaging probesbased on the disclosed polymer-conjugates are well suited for variousapplications (e.g., cancer detection and therapeutics) because thepolymers described herein i) integrate multimeric targeting ligands forreceptors in cancer cells to achieve high tumor specific uptake andretention, ii) contain multiple chelators to chelate multipleradiometals for enhanced specific activity and quantitative PET imaging,and iii) allow tunable hydrophilicity through minimal structural changesto increase plasma stability, prolong probe circulation in vivo, improvepharmacokinetics, and reduce immunogenicity. The modular aspect of these‘clickable’ polymer scaffolds allows for a library of derivatives to bequickly synthesized to tune their in vivo properties, as describedabove. In certain embodiments, a major advantage to these polymerscaffolds is the ability to easily change the peptide to change themolecular target and to change the chelator (e.g. DOTA instead of DFO)to change the radiometal. Changing the targeting peptide allows forthese systems to target a theoretically limitless number of moleculartargets, and changing the radiometal allows for PET or SPECT imagingwith a variety of radiometals with different emission properties andhalf-lives. Moreover, the disclosed polymer scaffolds also provideopportunities for therapy using isotopes such as ¹⁷⁷Lu and ⁹⁰Y.

In certain embodiments, one advantage of this modular polymer scaffoldis facile purification. For example, small peptide conjugates typicallyrequire HPLC purification and subsequent heating and solvent evaporationprior to formulation for injection. However, these polymer systems reachmolecular weights of 15-30 kDa, allowing for efficient purificationusing disposable size exclusion columns (PD-10) and disposablespin-filters (Amicon). This type of system is amenable for making kitformulations. As a result, these systems can be deployed in a hospitalradiopharmacy setting, unlike conventional small peptide conjugateswhich require HPLC purification by an expert radiochemist.

Synthesis of a DFO-Conjugated Polymer, Radiolabeling, and In VitroStability Tests

Polycarbodiimide polymers conjugated with the radiometal chelator,desferrioxamine B (DFO), a hexadentate ligand that chelates ⁸⁹Zr undermild conditions, fluorophore (IR650 dye), and PEG side chains (DFO-JBP1)were synthesized as shown in FIG. 17. ⁸⁹Zr PET tracer emits low energypositrons and exhibits a relatively long half-life (78.41 h) thatfacilitates high imaging resolution and allows imaging at multiple timepoints, for example as described by Deri et al J. Med. Chem. 2014. Thesynthesized DFO-conjugated polymer (DFO-JBP1) was efficiently (greaterthan 99%) radiolabeled with ⁸⁹Zr within 60 minutes at room temperature.The radiochemical purity was greater than 99.9% as determined fromradio-iTLC as is shown in FIG. 18A. Without having to be bound bytheory, negligible radiolabeling in control experiments with a similarpolymer without DFO-conjugation (mPEG-JBP2, FIG. 18B) suggests lack ofnon-specific labeling from polymer backbone. Table 3 shows that theconcentration dependent ⁸⁹Zr labeling of DFO-JBP1 showed nearquantitative radiolabeling with 100 μCi activities in 10 μg polymer.

TABLE 3 Concentration Dependent Radiolabeling Polymer Initial89Zr-Radiolabeling (%) weight (μg) activity (μCi) of DFO-JBP1 1 100 16.610 100 97.6 30 100 >99 100 100 >99 200 100 >99 400 100 >99

As shown in Table 3, the highest specific activities detected were 13mCi/mg polymers. Serum stability test on the ⁸⁹Zr-radiolabeled polymer(⁸⁹Zr-DFO-JBP1) in the presence of human blood serum showed 98-99%stability over seven days (FIG. 18C). The ⁸⁹Zr radiolabeling of thepolymer achieved high specific activities (e.g., 13 mCi/mg) enabling alow dose injection in mice.

PET Imaging and Biodistribution of Radiolabeled Polymer in Healthy Mice

⁸⁹Zr-labeled polymer without targeting ligands was i.v. injected (˜100μCi, ˜10 μg) into healthy mice (BALB/c) and imaged at three time points(2 h, 24 h, and 72 h) using PET (FIG. 19A). At 2 h post injection, themajority of radioactivity was detected in the blood, and at 24 hsignificant radioactivity was still detected in blood indicating a longcirculation time. Polymer derivatives with conjugated targeting peptidescan benefit from this long circulation time for higher tumor uptake.Overtime, most radioactivity was detected in the liver. The ex vivobiodistribution performed at 96 h post injection matched the trend withPET images with the highest activity detected in the liver (FIG. 19B).

Materials and Methods Chemicals

Reagents were purchased from Sigma-Aldrich, Milwaukee, Wis., AcrosOrganics, and Fisher Scientific, Fair Lawn, N.J., and used as received.Neutral silica gel (Ultrapure 60-200 μm, 60 Å, Acros Organics) was usedin column chromatography purification of monomers. Anhydrous andinhibitor-free tetrahydrofuran (THF) was used for click chemistry.

Material Characterization

NMR data were recorded on a Bruker Advance III Ultrashield Plus 500 MHzspectrometer at room temperature. The chemical shift values werereported relative to TMS (δ=0.00 ppm) as an internal standard. Fouriertransform infrared (FTIR) spectra were acquired using a Bruker OpticsTensor 27 FTIR spectrometer using ATR cell (Pike technologies).Wavenumbers in cm⁻¹ are reported for characteristic peaks. Allmanipulations for polymerization were done at room temperature inside anMBraun UNIlab drybox under inert atmosphere. High resolution massspectra (HRMS) were obtained on a Waters LCTPremier XE mass spectrometerby electrospray ionization. Size exclusion chromatography (SEC) wasperformed on a Viscotek GPCmax system (Malvern Instruments) equippedwith ViscoGEL columns (IMBMMW-3078 and I-MBLMW-3078 in series) connectedto a Viscotek TDA 305 triple detector array at 30° C. using THF as aneluent to determine relative molecular weights of the polymers.Polystyrene standards were used for the calibration of the instrument.Polymer samples were dissolved in the solvent system containing 0.12 Mdiethanolamine in THF, and the solutions were filtered through 0.45 μmPTFE filters prior to injection. The flow rate was 1.0 mL/min, andinjector volume was 100 μL. OmniSEC software was used to calculate themolecular weight. The polymer-SWCNTs zeta potential measurements werecarried out in a Zetasizer Nanoseries (Malvern Instruments).

Synthesis and Characterization of Compounds

Urea derivatives, monomers, and corresponding polymers (FIG. 20) wereprepared as described below (Budhathoki-Uprety, J.; Novak, B., Synthesisof Alkyne-Functionalized Helical Polycarbodiimides and their Ligation toSmall Molecules using ‘Click’ and Sonogashira Reactions Macromolecules2011, 44 (15), 5947-5954). Molar ratio of monomer to catalyst waslimited to 25:1 (Poly-1) or 32:1 (Poly-2) to obtain low molecular weightpolymers to improve aqueous solubility.

1-(3-ethynylphenyl)-3-propylurea, Compound 1

3-Amino phenylacetylene (1.0 g, 8.53 mmol, 1.0 equiv) was diluted inanhydrous dichloromethane (25 mL) and added to n-propylisocyanate (0.87g, 10.24 mmol, 1.2 equiv) in dichloromethane (10 mL), stirred at lowtemperature, and kept cold in an ice bath. The reaction mixture wasallowed to warm to room temperature followed by reflux overnight. Thesolvent was removed in a rotary evaporator and crude white solid waspurified by recrystallization in ethanol at 4° C. and dried to obtainwhite crystalline solid 1. 1H NMR (500 MHz, CDCl3, δ ppm): referenceTMS=0 ppm, δ=7.99 (s, 1H), 7.39 (s, 1H), 7.26 (d, 1H), 7.15-7.08 (m,2H), 6.02 (s, br, 1H), 3.11-3.07 (m, 2H), 2.99 (s, 1H), 1.45-1.38 (m,2H), 0.83 (t, J=7.5 Hz, 3H). 13C NMR (125 MHz, CDCl3, δ ppm): referenceCDCl3=77.23 ppm, δ=156.9, 139.4, 129.1, 126.6, 123.4, 122.8, 120.7,83.5, 77.3, 42.0, 23.4, 11.4. HRMS (ESI) [M+H]+m/z calcd for C12H15N2O,203.1184. found, 203.1187.

1-phenyl-3-(prop-2-yn-1-yl)urea, Compound 2

Propargyl amine (0.60 g, 10.89 mmol, 1.1 equiv) was diluted in anhydrousdichloromethane (20 mL) and added to phenylisocyanate (1.18 g, 9.90mmol, 1.0 mol equiv) in dichloromethane (20 mL), stirred at lowtemperature, and kept cold in an ice bath. The reaction mixture was thenallowed to warm to room temperature. A white precipitate resultedshortly after mixing with phenylisocyanate. The reaction mixture wasallowed to stir for 3 hours. The white solid was then separated andpurified by recrystallization in dichloromethane at 4° C. to obtainwhite crystalline solid 2. 1H NMR (500 MHz, DMSO-d6, δ ppm): referenceDMSO-d6=2.50 ppm, δ=8.56 (s, 1H), 7.40 (d, J=7.65 Hz, 2H), 7.23 (t,J=7.60 Hz, 2H), 6.91 (t, J=7.35 Hz, 1H), 6.45 (t, J=5.60 Hz, 1H), 3.90(dd, J=5.70 Hz, 2.45 Hz, 2H), 3.09 (t, J=2.45 Hz, 1H). 13C NMR (125 MHz,DMSO-d6, δ ppm): reference DMSO-d6=39.51 ppm, δ=154.7, 140.1, 128.6,121.4, 117.8, 82.1, 72.9, 28.7. HRMS (ESI) [M+H]+m/z calcd forC10H11N2O, 175.0871. found, 175.0863.

3-ethynyl-N-((propylimino)methylene)aniline, Compound 3

Triethyl amine (2.07 g, 20.51 mmol, 2.5 equiv) was added to a suspensionof dibromotriphenylphosphorane (4.15 g, 9.84 mmol, 1.2 mol equiv) indichloromethane (2 mL) and stirred at low temperature under inertatmosphere. After stirring the mixture for 5 minutes, compound 1 (1.66g, 8.20 mmol, 1.0 equiv) was added and the reaction mixture and stirreduntil completion. The dehydration of the urea derivative intocarbodiimide monomer was monitored by the formation of a very strongFTIR signal at ˜2120-2140 cm-1. Upon completion of the reaction, hexanewas added to precipitate side products. The monomer compound was thenextracted from solid by hexanes. Crude monomer was further purified bycolumn chromatography on silica gel using ethyl acetate:hexanes (1:2)and dried under reduced pressure to obtain 3 as a colorless oil. 1H NMR(500 MHz, CDCl3, δ ppm): reference TMS=0 ppm, δ=7.20 (m, 3H), 7.07-7.04(m, 1H), 3.39 (t, J=6.8 Hz, 2H), 3.07 (s, 1H), 1.73-1.1.69 (m, 2H), 1.01(t, J=7.4 Hz, 3H). 13C NMR (125 MHz, CDCl3, δ ppm): referenceCDCl3=77.23 ppm, δ=141.3, 129.5, 128.4, 127.1, 124.3, 123.3, 120.9,83.2, 77.7, 48.7, 24.9, 11.6. FTIR (thin film, cm-1): characteristicabsorption from terminal alkyne group and monomer; 3290 (terminalalkyne), 2123 (vs, carbodiimide).

N-((prop-2-yn-1-ylimino)methylene)aniline, Compound 4

The same procedure as described in the synthesis of compound 3 wasemployed. 1H NMR (500 MHz, CDCl3, δ ppm): reference TMS=0 ppm,δ=7.31-7.28 (m, 2H), 7.16-7.14 (m, 3H), 4.08 (d, J=2.45 Hz, 2H), 2.44(t, J=2.50 Hz, 1H). 13C NMR (125 MHz, CDCl3, δ ppm): reference TMS=0ppm, δ=139.5, 139.0, 129.4, 125.5, 124.0, 79.0, 73.5, 36.0. FTIR (thinfilm, cm-1): characteristic absorption from terminal alkyne group andmonomer; 3302 (terminal alkyne), 2119 (vs, carbodiimide). HRMS (ESI)[M+H]+ m/z calcd for C10H9N2, 157.0766. found, 157.0761.

Synthesis of Catalyst

The catalyst was synthesized and characterized following previouslydescribed procedure (Tang, H.; Boyle, P.; Novak, B., Chiropticalswitching polyguanidine synthesized by helix-sense selectivepolymerization using[(R)-3,3′-dibromo-2,2′-binaphthoxy](di-tert-butoxy)titanium(IV)catalyst. J. Am. Chem. Soc. 2005, 2136-2142).

Synthesis of Polymers

Polymers were synthesized following the reported procedure 1. Briefly,the catalyst, either neat or dissolved in chloroform (0.2 mL per 500 mgmonomer) was added to the monomer at room temperature and under inertatmosphere. The reaction mixture turned to dark red and solidified to anorange red solid. The polymerization process was monitored in FTIR bydisappearance of IR signals from carbodiimide (˜2140-2120 cm⁻¹) andformation of new IR absorption at ˜1620-1640 cm⁻¹ of the polymerbackbone. Upon completion of the polymerization (ca. 24 h), the solidwas dissolved in chloroform, precipitated in methanol, separated, anddried to obtain light yellow solid.

FTIR (thin film, cm-1): characteristic absorption from terminal alkynegroup and polymer backbone; 3304 (terminal alkyne C—H), 2123 (alkynetriple bond, C≡C), 1631 (imine in polymer backbone, C═N). 1H NMR (500MHz, CDCl3, δ ppm): reference TMS=0 ppm, δ=7.28-6.84 (br), 5.35-5.29(br), 4.37-4.20 (br), 3.14 (br), 2.07-0.75 (br).

FTIR (thin film, cm-1): characteristic absorption from terminal alkynegroup and polymer backbone, 3300 (terminal alkyne C—H), 2123 (w, alkynetriple bond, C≡C), 1624 (imine in polymer backbone, C═N). Mn=13, 346,PDI=1.29. 1H NMR (500 MHz, CDCl3, δ ppm): reference TMS=0 ppm,δ=7.15-6.49 (br), 3.45 (br), 3.19 (br), 3.03 (br), 2.53 (br) 1.01-0.70(br).

FIGS. 21A-21B show the FTIR spectra of polymers, Poly-1 and Poly-2,respectively.

Synthesis of Azides

Azide compounds, as shown in FIG. 22, were synthesized and characterizedfollowing literature procedures (Inverarity, I. A.; Hulme, A. N., Markedsmall molecule libraries: a truncated approach to molecular probedesign. Organic & Biomolecular Chemistry 2007, 5 (4), 636-643;Srinivasan, R.; Tan, L. P.; Wu, H.; Yang, P.-Y.; Kalesh, K. A.; Yao, S.Q., High-throughput synthesis of azide libraries suitable for direct“click” chemistry and in situ screening. Organic & BiomolecularChemistry 2009, 7 (9), 1821-1828; Budhathoki-Uprety, J.; Peng, L.;Melander, C.; Novak, B. M., Synthesis of Guanidinium FunctionalizedPolycarbodiimides and Their Antibacterial Activities. ACS Macro Lett.2012, (1), 370-374).

Coupling of Azides 5-7 to Poly-1 and Poly-2 Via ‘Click’ Chemistry toPrepare Poly-3-8.

To the stirring polymer solution in tetrahydrofuran under inertatmosphere, azide compound (2.0 mol equiv per alkyne unit), triethylamine or DBU (6.0 mol equiv per alkyne unit) and CuI (10 mol %) wereadded. The reaction mixture was stirred overnight under an argonatmosphere. Coupling of small molecules azides to alkyne side chains inpolymers was monitored by FTIR analysis. Upon completion of thereaction, the resulting polymer was washed with THF and/or diethylether, separated by filtration and dried under reduced pressure. FTIRanalysis of final polymers showed full conversion of all alkyne repeatunits in click reaction. Limited solubility of final polymers poseddifficulty in GPC measurements. Amine-Poly-6, Amine-Poly-8, andGuanidine-Poly-4 were acidified with a few drops of dilute HCl toincrease water solubility. Carboxy-Poly-7 was treated with a few dropsof saturated solution of NaHCO3. Acidic and basic polymer solutions werethen filtered through centrifugal filters (Amicon Ultracel®, MWCO 3K Da,Merck Millipore Ltd) to remove residual small molecules and washed withwater until free from free acid or base as tested with litmus paper. Thepolymers were then used to suspend SWCNTs.

Photoluminescence Excitation/Emission Contour Plots

Photoluminescence (PL) plots were constructed using a home-builtapparatus comprising of a tunable white light laser source, invertedmicroscope, and InGaAs nIR detector. The laser was a SuperK EXTREMEsupercontinuum white light laser source (NKT Photonics) with a VARIAvariable bandpass filter accessory capable of tuning the output 500-825nm with a bandwidth of 20 nm. A longpass dichroic mirror (900 nm) wasused to filter the excitation beam. The light path was shaped and fedinto the back of an inverted IX-71 microscope (Olympus) where it passedthrough a 20×nIR objective (Olympus) and illuminated a 200 μL nanotubesample in a 96-well plate (Greiner). Emission from the nanotube samplewas collected again by the 20× objective and diverted, via a long-passdichroic mirror (875 nm), matched to the f/# of the spectrometer usingseveral lenses, injected into an Isoplane nIR spectrograph (PrincetonInstruments) with a slit width of 410 μm, and dispersed by a grating of86 g/mm and 950 nm blaze wavelength. The light was collected by a PIoNIRInGaAs 640×512 pixel array (Princeton Instruments).

Excitation, emission, and wavelength corrections and calibrations wereperformed as follows. The power at each excitation wavelength wasmeasured at the objective with a PM100D power meter (Thorlabs) fromwhich a power spectrum was constructed and used to correct the emissionintensities for nonuniform excitation. A HL-3-CAL-EXT halogencalibration light source (Ocean Optics) was used to correct fornon-uniformities in the emission path arising from grating, detector,and lens inefficiencies. A Hg/Ne pencil style calibration lamp (Newport)was used to calibrate emission wavelengths ranging from 950-1350 nm.

Acquisition was conducted in semi-automated fashion controlled byLabview code which iteratively increased the excitation laser sourcefrom 491-824 nm in steps of 3 nm and saved the data in ASCII format.Using a center wavelength of 1135 nm, the emission spectra range was915-1354 nm with has a resolution of 0.7 nm. Background subtraction wasconducted using a well filled with DI H₂O. Following acquisition, thedata was processed with a Matlab code which applied the corrections fornon-uniform excitation and emission (as mentioned previously), createdthe contours with a Gaussian smoothing function, and output the figuresto be used for nanotube peak picking.

Topical Application of Polycarbodiimide-SWCNT Complexes on Human Skin.

Polycarbodiimide-SWCNT complexes (nanotube concentration in aqueoussuspension: 70-90 mg/L) were deposited onto normal human skin afterharvesting from patients during Moh's surgery. Normal skin at theperiphery of the tumor was used; the tumor tissue was discarded. After atwo-hour exposure to nanotubes, the skin surface was wiped off to removeunabsorbed nanotubes. The skin samples were then microtomed into 5 mmthick slices and imaged under 730 nm excitation.

1. A suspension of helical-polymer-encapsulated carbon nanotubes,wherein the helical polymer is a polycarbodiimide.
 2. The suspension ofclaim 1, wherein the carbon nanotubes are single-walled carbon nanotubes(SWCNTs).
 3. The suspension of claim 1, wherein the helical polymercomprises a clickable polymer scaffold.
 4. The suspension of claim 1,wherein the polycarbodiimide comprises one or more monomeric speciesselected from the group consisting of


5. The suspension of claim 1, wherein the suspension is an aqueoussuspension.
 6. The suspension of claim 1, wherein at least a pluralityof the helical-polymer-encapsulated carbon nanotubes in the suspensionare in van der Waals contact at a center-to-center distance betweenadjacent nanotubes sufficient to exhibit inter-nanotube Försterresonance energy transfer (INFRET).
 7. The suspension of claim 6,wherein the center-to-center distance is from 1 nm to 4 nm.
 8. Thesuspension of claim 6, wherein the dispersedhelical-polymer-encapsulated carbon nanotubes in van der Waals contactare not irreversibly bound.
 9. The suspension of claim 6, comprising (i)a first set of nanotubes each encapsulated by a helical polymer havingat least a first substituent functional group, and (ii) a second set ofnanotubes each encapsulated by a helical polymer having at least asecond substituent functional group, wherein the first substituentfunctional group and the second substituent functional group imbue thefirst and second sets of encapsulated nanotubes with sufficiently strongcoulombic attraction to each other to form reversible fluorescentaggregates in the suspension.
 10. The suspension of claim 1, wherein thehelical polymer comprises functional side chains.
 11. The suspension ofclaim 10, wherein the functional side chains comprise one or moremembers selected from the group consisting of a primary amine, acarboxylic acid, a guanidine group, an oligoethylene glycol, amethoxy-polyethylene glycol (PEG), a hydroxyl-PEG, a folic acid, atrimethoprim, a peptide, an alkyne peptide, an adenosine triphosphate(ATP) peptide, and an opioid.
 12. The suspension of claim 1, wherein thehelical polymer comprises one or more aromatic groups incorporated inits monomer substituents.
 13. The suspension of claim 12, wherein theone or more aromatic groups promote multi-valent π-π interactionsbetween the polymer and the graphitic sidewall of the carbon nanotubes.14. The suspension of claim 1, wherein the functional side chainscomprise a targeting group.
 15. A biomolecular imaging probe and/orsensor comprising the suspension of claim
 1. 16. An imaging methodcomprising: administering the suspension of claim 1 to a biologicalsample; exposing the biological sample comprising the administeredsuspension to excitation light; and detecting light emitted bysuspension or fluorescent aggregates formed by one or more components ofthe suspension.
 17. The method of claim 16, further comprisingdisrupting the fluorescent aggregates to reverse the emission of light.18. The method of claim 17, further comprising alternating betweencycles of light emission and no light emission by re-aggregating anddisrupting, respectively, the fluorescent aggregates for high resolutionbiomolecular imaging.
 19. The method of claim 16, wherein the detectingstep comprises obtaining images of cellular nuclei of the biologicalsample.
 20. A method of treating a disease or disorder, the methodcomprising administering the suspension of claim 1 to a subject, whereinthe functional side chains of the helical polymer comprises atherapeutic.
 21. The method of claim 20, wherein the therapeuticcomprises an opiate.
 22. A method for pretargeted radioimmunotherapy(PRIT), the method comprising administering a polycarbodiimidefunctionalized with an antibody, labeled with a radionuclide.
 23. Themethod of claim 22, wherein the radionuclide comprises a metalliclanthanide.
 24. The method of claim 22, wherein the radionuclide isattached to the polycarbodiimide via a chelator.
 25. The method of claim22, wherein the radionuclide comprises ⁸⁹Zr.
 26. The suspension of claim1, wherein the suspension is a stable suspension in aqueous solution orin serum. 27-71. (canceled)